1. Field of the Invention
This invention relates to the field of virology and immunology. Particularly, but not exclusively, it relates to a method of inducing an immune response, and a substance based on the amino terminal end of the matrix protein (p17MA) and covalent binding site for myristate (SEQ ID NOS: 1-3) of the HIV virus for achieving the same.
2. Description of the Related Art
Introduction
Human Immunodeficiency Virus (HIV) is a retrovirus within the slow or Lentivirus group, and is the cause of Acquired Immunodeficiency Syndrome (AIDS). Like many enveloped viruses, HIV fuses the viral and cellular membrane, leading to infection and viral replication. Once it has fused to a host cell, HIV transfers its genome across both the viral and cellular membranes into the host cell.
HIV uses its RNA as a template for making complementary viral DNA in target cells through reverse transcription. Viral DNA can then integrate into the DNA of an infected host. HIV infects cells having surface CD4, such as lymphocytes and macrophages, and destroys CD4 positive helper T lymphocytes. (CD4 represents a Cluster of Differentiation Antigen no. 4 that is part of both Th1 and Th2 cells.) Cell membrane molecules are used to differentiate leukocytes into various effector subsets. In general, four types of cell membrane molecules also known as cluster of differentiation (CD) have been delineated. Type I and II are transmembrane proteins (TPs) with opposite polarity crossing the plasma membrane. Type III TPs crosses the plasma membrane several times and therefore may form pores or channels. Type IV TPs are linked to glycosylphosphatidylinositol (GPI). CD4 is a type I transmembrane protein expressed on a variety of cells including helper/inducer T cells, monocytes, macrophages and antigen presenting cells.
This process relies in part on fusion protein, which is a component of the gp41 glycoprotein. The F protein structure is protease resistant. (Weissenhorn, Nature Vol. 387, pp. 426-430 (1997)) Using X-ray crystallography the three dimensional features of the F protein have been delineated.
The outer membrane proteins, gp41 and gp120, of the HIV virus are non-covalently bound to each other. On the surface of the HIV virion gp120 and gp41 are assembled into a trimeric unit. Three molecules of gp120 are assimilated with three gp41 molecules.
The gp120 molecule binds to a CD4 receptor on the surface of helper T cells as well as macrophages and monocytes. This binding is characterized by a high affinity between the two molecules. High sialic acid content on the surface of the virus reduces the threshold binding energy needed to overcome repulsive electrostatic forces. (Sun, 2002) Membrane fusion of an HIV particle to a target host cell may thus be considered to involve the following steps:                1. interaction of viral bound CypA with host/cellular heparin.        2. viral attachment to target cell via CypA/heparin interaction.        3. gp120 binding to the CD4 molecule of the target cell. This process requires coreceptor proteins also known as chemokine receptors (CCR5 for T cells and CXCR4 for macrophages). The virus then begins to fuse with the cell, producing structural or conformational changes and exposing other receptors;        4. conformational three dimensional and/or tertiary structure changes of the gp120 molecule exposing the fusion domain or F protein of gp41;        5. dissociation of the gp120 from the gp41 molecule as a result of the conformational change and the shedding of gp120;        6. folding of gp41 upon itself before piercing the plasma membrane of the target cell        7. unfolding of the F protein; and        8. fusion of the membranes of the viral particle and host cell.The insertion of the fusion peptide disrupts the integrity of the lipids within the targeted host cell membrane. F protein links the viral and the cellular membranes, such that upon unfolding of the fusion protein, the plasma membrane of the target cell and the viral membrane are spliced together.        
The viral membrane of HIV is formed from the plasma membrane of an infected host cell when the virus buds through the cell's membrane. Thus, the envelope includes some of the lipid and protein constituents of the host cell. (Stoiber, 1996) (Stoiber, 1997) Some enveloped viruses use spike proteins, etc., to mimic the host molecules in order to bind to target cell receptors and to enter other target cells. However, these spikes can also be antigenic surfaces for immune system recognition and viral destruction. HIV protects itself against immune challenge (humoral and CD8 mediated) by the host. In addition to the variability of conformational changes, gp120 provides other surface features that disguise it from immune detection and attack, such as a coating of glycoproteins, covalently bound sialic acid residues, or steric occlusion. (Haurum, 1993) In short, HIV activates a variety of immune responses to its own advantage.
The core of the HIV virion functions as a command center. Inside an HIV virion is a capsid composed of the viral protein p24 (CA). The capsid also holds two single strands of RNA, each strand of which provides a copy of HIV's nine genes, which encode 15 proteins. Of the nine genes, three (gag, pol and env) are considered essential. Six additional genes are also found within the 9-kilobase pair RNA genome (vif, vpu, vpr, tat, rev, and nef). More specifically, the env gene holds the information or code for creation of gp160, which breaks down into gp120 and gp41. Likewise the gag gene encodes the matrix (p17 or MA), capsid (p24 or CA), nucleocapsid (p9, p6 or NC). The pol gene provides the genetic information for the virus to produce the reverse transcriptase enzyme as well as the integrase enzyme and RNAseH enzyme. The other six genes are regulatory, and control the mechanisms of infection and replication (tat, rev, nef, vif, vpr and vpu). Among other things, the nef gene holds information for efficient replication, while vpu holds information regulating the release of new viral particles from the infected host cell. Ultimately, in order for HIV to infect a target cell, it must inject the HIV genetic material into the target cells cytoplasm.
As noted above, the nef gene is believed to aid efficient replication of HIV. The creation of a new virus particle occurs at the host cell's membrane. Nef appears to affect an infected cell's environment in a way that optimizes replication. Viral proteins collect near the host cell's membrane, bud out within the membrane, and break away. These proteins are the three structural proteins (gp160, gp120, gp41) plus two other internal precursor polyproteins (Gag and the Gag-Pol). The Gag-Pol protein brings two strands of the positive RNA into the bud, while protease cuts itself free. After the virus has budded, protease cuts itself free and cuts up the rest of the proteins in Gag or Gag-Pol, releasing the various structural proteins and reverse transcriptase. The viral proteins are not functional until they are separated by the protease. Thus, protease is responsible for cleavage of Gag-Pol and the smaller Gag polyprotein into structural proteins. Released proteins p24, p7 and p6 form a new capsid, while at the base of the lipid membrane is p17. In this process, gp160 breaks down into gp120 and gp41 by a host enzyme.
The gag gene gives rise to a 55-kilodalton (kD) Gag precursor protein, also called p55 (Pr55gag), which is expressed from the unspliced viral messenger RNA (mRNA). During translation, the N terminus of the p55 is myristylated, triggering its association with the cytoplasmic aspect of cell membranes. The membrane-associated Gag polyprotein recruits two copies of the viral genomic RNA along with other viral and cellular proteins that trigger the budding of the viral particles from the surface of an infected cell. After budding, p55 is cleaved by the virally encoded protease (a product of the Pol gene), during the process of viral maturation into four smaller proteins designated MA (matrix or p17), CA (capsid or p24) and NC (nucleocapsid or p9 and p6.) (Cohen, P. T., et al., The AIDS Knowledge Base, 149 (1999)) Thus, the HIV core contains four proteins, including p17. In summation, the HIV virus is encoded by three large genes encoding structural and enzymatic peptides (gag, pol and env) and six smaller regulatory genes (vif, vpu, vpr, tat, rev and nef). (Sande, Merle A., et al., The Medical Management of AIDS, Ch. 2 (6th ed. 1999))
Pr55gag (the polypeptide encoded by the gag gene) is cleaved by viral protease to generate four large and two small peptides. From N to C terminus the following proteins are proteolytically produced: matrix (p17MA), capsid (p24CA), nucleocapsid (p7NC), and p6. The two small peptides include p2 located between p24CA and P7NC and p1 located between p7NC and p6. The matrix protein assembles inside the viral lipid bilayer and stabilizes it. (Zhou, Wenjun, et al., J. of Virology, pp 8540-8548 (December 1996))
Two critical steps in HIV viral replication are controlled by the matrix protein and the larger polyprotein precursor Pr55gag. (1) The nuclear targeting signal of the matrix protein. (2) The strong localizing signal to the cell plasma membrane of Pr55gag (Lee, Young-Min, et al., J. of Virology, pp 9061-9068 (November 1998))
The Pr55gag localizing signal can differentiate between the membranes encompassing cellular organelles and the plasma membrane. The key to the specificity of plasma membrane binding is conferred by a combination of a basic residue rich domain (amino acids 17-31) and the presence of an N-terminal myristoyl moiety. The 14 carbon fatty acid myristate is cotranslationally attached to the N-terminus of the HIV Pr55gag. This plasma membrane targeting of Pr55gag is essential for viral assembly and budding. (Zhou, 1996)
The active nuclear transport of the preintegration complex of HIV disease is controlled by two viral proteins, p17MA and Vpr. After fusion of the viral membrane to the target cell membrane the matrix protein becomes detached from the inner aspect of the lipid bilayer and several matrix molecules with viral RNA cross the nuclear membrane and enter the nucleoplasm. Therefore HIV-1 can infect non-dividing cells and is not dependent on the disintegration of the nuclear envelope which occurs during mitosis. Most viruses are not capable of infecting non-dividing cells. (Zhou, 1996)
The differential membrane binding of Pr55gag and MA is due to a myristoyl switch. Myristate is covalently bound to the N-terminal glycine amino acid of the MA protein. (Ono, Akira, et al., J. of Virology,” pp 4136-4144 (May 1999)) Upon cleavage of the Pr55gag by viral protease the myristate moiety inserts into a preexisting cavity of the MA molecule. This change in the three dimensional structure of the MA molecule occurs as a result of altered conformation in amino acids 9-11 (serine, glycine, glycine) Therefore the MA molecule has less electrostatic force holding it to the lipid bilayer than its larger precursor Pr55gag. Disruption of the viral plasma membrane upon fusion to a target cell destabilizes the matrix complex allowing for nuclear localization to occur. The flipping of the myristate moiety in and out of a protected cleft is known as the myristoyl switch and is found in other proteins including ADP ribosylation factor (ARF), recoverin and c-Abl. Current estimates of myristoylated proteins in the human genome approximate 0.5%. (Resh, Marilyn D., “A myristoyl switch regulates membrane binding of HIV-1 Gag,” Proc. Natl. Acad. Sci., Vol 101 (2) 417-418 (Jan. 13, 2004))
Myristylated proteins are covalently attached to myristic acid [tetradecanoic acid CH3 (CH2)12 COOH]. Myristic acid is a carboxylic acid. Carboxylic acid molecules are polar and like alcohol molecules can form hydrogen bonds with each other and with other kinds of molecules. Myristic acid is virtually insoluble in water but is highly soluble in lipids explaining in part the plasma membrane localizing signal inherent in the MA molecule.
Fatty acid components of proteins can serve as regulated targeting devices. (Tedeschi, Henry, Cell Physiology Molecular Dynamics, ch. 4 (2003)) The carboxyl end of several myristylated proteins provide hydrophobic anchors used in protein targeting and in signal transduction. The fatty acid site may attach via hydrophobic interactions to the phospholipid bilayer. Myristic acid is added cotranslationally (while the protein is being synthesized) on terminal glycine amino acids.
The MA polypeptide (p17) is derived from the N-terminal, myristylated end of p55. Most MA molecules remain attached to the inner surface of the virion lipid bilayer, stabilizing the particle. A subset of MA is recruited inside the deeper layers of the virion where it becomes part of the complex which escorts the viral DNA to the nucleus after fusion of the viral and host membranes have occurred. These MA molecules facilitate nuclear transport of the viral genome because a karyophilic signal on MA is recognized by the cellular nuclear import machinery. This is important because this allows HIV to infect non-dividing cells, such as macrophages, which is an unusual property for a retrovirus. (Cohen, P. T., et al., The AIDS Knowledge Base 149 (1999))
Most HIV vaccines, however, use portions of envelope glycoproteins (gp160, gp120, and gp41) in an attempt to induce production of neutralizing antibodies against the envelope spikes of the virus. (Johnston, et al., 2001) Some have been successful in producing high titers of neutralizing antibodies. The thought behind this approach is that the antibodies that bind to these glycoproteins would neutralize the virus and prevent infection. A functioning immune system could then activate the complement system, which would cascade to lysis and destroy the virus. The complement system is a series of circulating proteins that “complements” the role of antibodies. The components of the complement system are activated in sequence or turn, which is the complement cascade. The conclusion of complement is a protein complex, the Membrane Attack Complex (MAC) that seeks to attach to an invading organism's surface and to destroy it by puncturing its cell membrane.
Immune Response
Thus, a primary effect of HIV is to deplete the CD4 T+ cells, which lowers overall immune activity. As described above, HIV infection centers on CD4 T+ cells, but it also infects B cells, blood platelets, endothelial cells, epithelial cells, macrophages, etc. As CD4 T+ cells are depleted, the B cell response becomes deregulated. Hypergammaglobulinemia with ineffective antibodies characterizes HIV progression. Further, cytotoxic CD8 T cells are rendered incompetent and are unable to recognize and attack viral infection. This is due in part to transfection of uninfected CD8 cells with the tat protein manufactured in infected CD4 cells.
The CD4 T+helper (Th) cells produce cytokines and can be grouped into either Th1 cells or Th2 cells. The Th1 cells promote cell-mediated immunity while Th2 cells induce humoral immunity. The cytokines are chemical messengers or protein attractants that regulate immunologic responses. The depletion of CD4+ helper cells in HIV disease results in reduced synthesis of certain cytokines and enhanced synthesis of others. Cytokine disregulation depresses the activity of the natural killer cells and macrophages. Further, the loss of interleukin-2 slows the clonal expansion and activation of mature T cells.
Different viral traits augment or diminish cell mediated and humoral response. In some strains and phases of progression, HIV may be characterized as a failure of Th1 response, accompanied by overactive but ineffective Th2 response. The balance between Th1 and Th2 immune response appears to depend in part on the HIV strain(s) and in part on the genetic milieu of the infected animal. For example, long term nonprogressors mount an effective Th1 response to HIV disease. (Pantaleo, 1995)
An immunogenic compound directed to creating a balanced immune response and strengthening or reinforcing the type of immune response suppressed by a particular virus would be of value. (Hogan, 2001)
Cellular Response
HIV appears to trigger an initially strong cellular immune response that is not maintained over time and ultimately fails to control the infection. (McMichael, 2001)
CD8 cytotoxic T-cells (Tc) recognize a cell presenting a foreign antigen by MHC (Major Histocompatibility Complex) class 1 molecules on the surface, and attack it. CD4 helper cells (Th) stimulate macrophages that have ingested a viral microbe to kill the microbe. The cytokines or interleukins produced by the CD4 cells determine in part whether the immunologic response to a pathogen is primarily TH1 or TH2 driven. In some infections CD4 cells produce interleukin-4 and interleukin-5, which select for B-cells. B cells present antigen complexed with MHC class II molecules. In other infections CD4 cells produce IL-2 which select for cytotoxic T cells. This division or restriction of functions in recognizing antigens is sometimes referred to as MHC restriction. MHC class I generally presents endogenously synthesized antigens, such as viral proteins, while MHC class II generally presents extracellular microorganisms or antigens such as bacterial or viral proteins which have been phagocytosed by antigen presenting cells. The antigen presenting cells then bind the antigen with MHCII protein on its surface. The CD4 cell interacts with this antigen through its T cell receptor and becomes activated. This contributes to the ineffectiveness of inactivated vaccines to produce Tc cytotoxic response. (Levinson, 2002)
As noted above, T cells mediate cellular response. The antigen presenting cells, along with MHC molecules (or Human Leukocyte Antigen—HLA) present peptide portions of HIV antigens (or epitopes) to their respective T cells, triggering T cell response. The type of epitope presented to a T cell depends on the type of HLA molecule (e.g., HLA A, B, C, DR, DQ, DP) and the amino acid in the peptides. Genetic limitations in HLA molecules or mutant epitopes may lead to epitope escape and HIV persistence. (McMichael, 2001) As noted above, Th cells produce cytokines for general (i.e., Th 1 and Th2) immune response, but in the case of HIV this is suppressed by infection of the Th cells. HIV specific Th cells that respond to HIV antigens are eventually infected and destroyed or suppressed. This leads to a secondary effect on cytotoxic T cells. Cytotoxic T cells demonstrate a variety of antiviral activities (such as the production of performs, granzymes, FasL and cytokines), after recognizing and attacking foreign antigens on infected cells that are bound by MHC class I molecules. HIV can reduce the expression of HLA class I molecules in infected cells, reducing the ability of cytotoxic T cells to recognize and attack the infected Th cells. Further, the infection and depletion of Th cells disrupt the ability of cytotoxic T cells to mature and to address mutant virions. (McMichael, 2001) Typically, in a viral infection the cytotoxic T cells eliminate or suppress the virus. But HIV counters cellular immune response by infecting immune cells and impairing the response of Th cells and cytotoxic T cells.
Thus, an immunogenic compound that stimulated Th1 activity would promote favorable immune response against HIV.
Humoral Response
The humoral arm of the immune system consists of B cells that, upon stimulation, differentiate into antibody producing plasma cells. The first antibodies to appear are IgM, followed by IgG in blood, or IgA in secretory tissues. A major function of these antibodies is to protect against infectious disease and their toxins. Antibodies not only neutralize viruses and toxins, but also opsonize microorganisms. Opsonization is a process by which antibodies make viruses or bacteria more easily ingested and destroyed by phagocytic cells. Phagocytic cells include both polymorphonuclear neutrophils (PMNs) and tissue macrophages. PMNs comprise about 60% of the leukocytes in the blood of an uninfected patient. The number of PMNs and tissue macrophages may increase or decrease with certain infectious disorders. For example, typhoid fever is characterized by a decrease in the number of leukocytes (i.e., leukopenia). Both PMNs and macrophages phagocytose consume bacteria and viruses. PMNs do not present antigen to helper T cells, whereas macrophages and dendritic cells do.
Phagocytosis includes (1) migration, (2) ingestion, and (3) killing. Tissue cells in the infected area produce small polypeptides known as chemokines. The chemokines attract PMNs and macrophages to the site of an infection. Then the bacteria are ingested by the invagination of the PMN cell membrane around the bacteria to form a vacuole or phagosome. This engulfment or opsonization is enhanced by the binding of IgG antibodies (opsonins) to the surface of the bacteria. The C3b component of the complement system enhances opsonization. (Hoffman, R. Hematology Basic Principles and Practice Ch. 37 (3rd ed. 2000)) The cell membranes of PMNs and macrophage have receptors for C3b and the Fc portion of IgG.
With engulfment, a metabolic pathway known as the respiratory burst is triggered. As a result two microbicidal agents, the superoxide radical and hydrogen peroxide are produced within the phagosomes. These highly reactive compounds often called reactive oxygen intermediates are synthesized by the following chemical reactions:O2+e- ->O2—2O2—+2H+->H2O2 (Hydrogen peroxide)+O2 
The first reaction reduces molecular oxygen to form the superoxide radical, which is a weak microbicide. The second reaction, which is catalyzed by the enzyme superoxide dismutase within the phagosome, produces hydrogen peroxide. In general, hydrogen peroxide is a more effective microbicide than the superoxide radical. The respiratory burst also produces nitrous oxide (NO), another microbicide. NO contains a free radical that participates in the oxidative killing of ingested viruses and bacteria phagocytosed by neutrophils and macrophages. The NO synthesis within the phagosome is catalyzed by the enzyme NO Synthase, which is induced by the process of phagocytosis.
The killing of the organism within the phagosome is a two step process that consists of degranulation followed by the production of hypochlorite ions, which is the most effective of the microbicidal agents. Two types of granules are found within the cytoplasm of the neutrophils or macrophages. These granules fuse with the phagosome to form a phagolysosome. The contents of the granules are then emptied. These granules are lysosomes that contain a variety of enzymes essential to the killing and degradation. Two types of lysosomal granules, which are differentiated by their size, have been identified. The larger lysosomal granule, which constitutes about 15% of the total, contains several enzymes including myeloperoxidase, lysozyme, and other degradative enzymes. The remaining 85% are smaller granules, which contain lactoferrin and other degradative enzymes, such as proteases, nucleases, and lipases. The actual killing or destruction of microorganisms occurs by variety of mechanisms, some oxygen-dependent and some oxygen-independent. The most important oxygen-dependent mechanism is the production of the hypochlorite ion catalyzed by myeloperoxidase:Cl−+H2O2->CIO+H2O
Antibodies are glycoproteins, composed of light (L) and heavy (H) polypeptide chains. The simplest antibody has a “Y” shape and consists of four polypeptides: 2H-chains and 2 L-chains. Disulfide bonds link the four chains. An individual antibody molecule will have identical H- and identical L-chains. L- and H-chains are subdivided into two regions: variable and constant. The regions have segments or domains, which are three-dimensionally folded and repeating. An L-chain consists of one variable (V1) and one constant (C1) domain. Most H chains consist of one variable (VH) and three constant (CH) domains. The variable regions are responsible for antigen (virus, bacteria, or toxin) binding. The constant regions encode several necessary biologic functions including complement fixation and binding to cell surface receptors. The complement binding site is located in the CH2 domain.
The variable regions of both L- and H-chains have three highly variable (or hypervariable) amino acids sequences at the amino-terminal portion that makes up the antigen binding site. Only 5-10 amino acids in each hypervariable region form this site. Antigen-antibody binding involves electrostatic forces and van der Waals' forces. In addition, hydrogen and hydrophobic bonds are formed between the antigen and hyper-variable regions of the antibody. The specificity or “uniqueness” of each antibody is in the hyper-variable region; the hyper-variable region is the thumbprint of the antibody.
The amino-terminal portion of each L-chain participates in antigen binding. The carboxy-terminal portion contributes to the Fc fragment. The Fc fragment (produced by proteolytic cleavage of the hinge region of the antibody molecule separating the antigen binding sites from the rest of the molecule or the Fc fragment) expresses the biologic activities of the constant region, specifically complement fixation. The H-chains are distinct for each of the five immunoglobulin classes. The heavy chains of IgG, IgA, IgM, IgE and IgD are designated γ, α, μ, ε and δ respectively. The IgG immunoglobulin class opsonizes microorganisms; thus, this class of Ig (immunoglobulin) enhances phagocytosis. (Hoffman, Ronald, et al., Hematology Basic Principles & Practice, ch. 36 & 39 (3rd ed. 2000))(Levinson, Warren, Medical Microbiology & Immunology, Ch. 59 & 63 (7th ed. 2002)) Receptors for the γ H-chain of IgG are found on the surface of PMNs and macrophages. IgM does not opsonize microorganisms directly because there are no receptors on the phagocyte surface for the μH-chain. IgM does, however, activate complement, and the C3b protein can opsonize because there are binding sites for C3b on the surface of phagocytes. (Levinson, 2002) IgG and IgM, are able to initiate complement cascade. In fact, a single molecule of IgM can activate complement. Activation of complement by IgG requires two cross-linked IgG molecules (IgG1, IgG2, or IgG3 subclasses, IgG4 has no complement activity). A variety of non-immunologic molecules, such as bacterial endotoxin, can also activate the complement system directly.
The complement system consists of approximately twenty proteins that are normally in serum. The term “complement” indicates how these proteins complement or augment other components in the immune system, such as antibodies and immunoglobulin. Complement cascade has three important immune effects: (1) lysis of microorganisms; (2) generation of mediators that participate in inflammation and attract PMNs; and (3) opsonization.
Complement cascade occurs via one of three paths: (1) classic; (2) lectin; and (3) alternative. (Prodinger, Wm., et. al., Fundamental Immunology, Ch. 29 (1998)) These pathways are diagrammed in FIG. 1. The dashed line shows that proteolytic cleavage of the molecule at the tip of the arrow has occurred. A line over a complex indicates that it is enzymatically active. Although the large fragment of C2 is sometimes interchangeably labeled C2a or C2b, for convention, here small fragments are designated as “a,” and all large fragments as “b.” Hence, the C3 convertase is C4b,2b. Note that proteases associated with the mannose-binding lectin cleave C4 as well as C2. Each of these pathways leads to the creation of the Membrane Attack Complex (MAC).
With the antibody attached to a specific component of a virus or bacteria, the MAC is able to perforate the microorganism's protective cover and allow blood plasma and electrolytes to enter the microorganism, and at the same time provide a means for egress of the microorganism's internal structural components.
In the classic pathway, antigen-antibody complexes activate C1 to form a protease, which cleaves C2 and C4 to form a C4b,2b complex. C1 is composed of three proteins: C1q, C1r, and C1s. C1q is composed of 18 polypeptides that bind to the Fc portion of IgG and IgM. Fc is multivalent and can cross-link several immunoglobulin molecules. C1s is a proenzyme that is cleaved to form an active protease. Calcium is required as a cofactor in the activation of C1. Further, activation of C1 requires multi-point attachment of at least two globular heads of C1q to the Fc domains of IgG and/or IgM. The changes induced in C1q on binding multiple Fc immunoglobulins is transmitted to the C1rs subunits, resulting in proteolytic autoactivation of the C1r dimer, which then proteolytically activates or cleaves C1s. As seen above, activated C1s possesses the catalytic site for proteolytic splicing of C4 and C2. An enzyme complex, C4b,2b, is produced. This functions as a C3 convertase, which cleaves C3 molecules into two fragments, C3a and C3b. C3b forms a complex with C4b and C2b, producing a new enzyme, (C4b,2b,3b) which is a C5 convertase.
In the lectin pathway, mannan-binding lectin (MBL, or mannose-binding protein) binds to the surface of microbes expressing mannan. MBP is a C-type lectin in plasma that has a structure similar to that of C1q, and binds to C1q receptors enhancing phagocytosis. Mannose is an aldohexose found on the surface of a variety of microorganisms. The first component of the lectin pathway is designated mannose (or mannan) binding protein (MBP). A C-terminal carbohydrate recognition domain has affinity for N-acetylglucosamine and confers the capacity for MBP to directly opsonize microorganisms expressing mannose-rich surface coats. In the blood, MBP circulates as a stable complex with a C1r-like proenzyme and a C1s-like proenzyme (designated MBP-associated serine protease, or MASP-1 and MASP-2 respectively). The MBP-MASP-1, MASP-2 complex binds to the appropriate carbohydrate surface. This results in conformational change in the MBP protein which leads to auto-activation of MASP-1 by internal peptide cleavage converting MASP-1 to an active serine protease. Like C1r, active MASP-1 cleaves MASP-2 activating it. Active MASP-2 exhibits the capacity to proteolytically activate both C4 and C2 to initiate assembly of the C4b,2b (C3 convertase) enzyme complex. As with the classic pathway, this leads to the production of C5 convertase.
In the alternative pathway many unrelated cell surface structures, such as bacterial lipopolysaccharides (endotoxin), fungal cell walls, and viral envelopes, can initiate the process by binding to C3(H2O) and factor B. This complex is cleaved by a protease, factor D, to produce C3b,Bb, which acts as a C3 convertase to generate more C3b. In contrast to the sequential enzyme cascade of the classical pathway, the alternative pathway uses positive feedback; the principal activation product, C3b, acts as a cofactor for C3b,Bb, which is also responsible for its own production. Thus, the alternative pathway is continuously primed for explosive C3 activation. The rate of C3 activation is governed by the stability of the C3b,Bb enzyme complex. Proteolysis of factor B by factor D produces a small fragment (Ba) and a large fragment (Bb). The larger Bb fragment combines with either C3(H2O) or C3b. Through a catalytic site in Bb, the complex C3(H2O),Bb can proteolytically convert C3 to C3a and C3b. Nascent C3b generated by this mechanism is capable of binding additional factor B. Therefore the alternative complement pathway has at least two positive feedback loops enhancing the production of C3b. As shown in FIG. 1, this route also leads to the production of C5 convertase.
For each pathway the C5 convertase (C4b,2b,3b or C3b,Bb, C3b) cleaves C5 into C5a and C5b. C5b binds to C6 and C7, to form a complex that interacts with C8 and C9, ultimately producing MAC (C5b,6,7,8,9). (Hoffman, 2000)
Regardless of which complement pathway is activated, the C3b complex is a central molecule for complement cascade. Immunologically C3b fulfills three roles:                1. sequential combination with other complement components to generate C5 convertase, the enzyme that leads to production of MAC (C5b,6,7,8,9);        2. opsonization of microorganisms. Phagocytes have receptors for C3b on their cell surface.        3. binding to its receptors on the surface of activated B cells, which greatly enhances antibody production. (Parham, Peter, The Immune System, ch. 7 (2nd ed. 2004))The humoral response includes certain regulators of this system, such as Complement Factor H, that are vulnerable to exploitation by HIV. Any microorganism with the capacity to limit the activity of complement cascade could theoretically protect itself against the humoral arm of the immune system. (Stoiber, Heribert, Role of Complement in the control of HIV dynamics and pathogenis, Vaccine 21: S2/77-S2/82 (2003)) Thus, the complement cascade is an Achilles heel of the humoral arm.HIV Interaction with Humoral Response        
Retroviruses can activate the complement system in the absence of antibodies. (Haurum, J., AIDS, Vol. 7(10), pp. 1307-13 (1993)) Complement activation by HIV envelope glycoproteins has been found to be mediated by the binding of MBP to carbohydrates on natural envelope protein produced in virus-infected cells, as well as on glycosylated recombinant envelope proteins. (Haurum, John, AIDS, Vol. 7(10), pp. 1307-13 (1993)) (Speth, C., Immunology Reviews, Vol. 157, pp. 49-67 (1997)) Activation of the classical complement pathway and lectin pathway by retrovirus envelopes can be initiated by the binding of MBP to carbohydrate side chains of envelope glycoproteins. The transmembrane protein of HIV-1, gp41, has been shown to be non-covalently associated with gp120. Complement component, C1q, also binds to gp41. In the cell-external part (ectodomain) of gp41, three sites (aa 526-538; aa 601-613 and aa 625-655) bind both gp120 and C1q. Thus, C1q and gp120 are both structurally and functionally homologous. The interaction between gp41 and C1q is calcium dependent unlike the association of gp41 and gp120 which is calcium independent.
HIV triggers the classical and lectin pathway in an antibody-independent manner which leads to the infection of complement receptor-positive cells by HIV. The binding of C1q to gp41 may facilitate infection in different ways. C1q binds directly to HIV-infected cells that are also infected with HIV-1. C1q retains its ability to bind to the C1q receptor, also known as the collectin receptor. Further, gp41 interacts directly with C1q anchored on the plasma membrane of macrophages. In both cases, HIV has the opportunity for C1q-mediated CD4 independent contact with cells.
The homology of gp120 and C1q raises the possibility that gp120 may interact directly with the C1q receptor, and thereby facilitate the entry of HIV into macrophages in a CD4-independent manner. (Stoiber, Heribert, European Journal of Immunology, Vol 24, pp. 294-300 (1994)) Antibodies to gp120 are able to cross react with C1q and may be responsible, at least in part, for the significantly low C1q concentration in HIV-1 patients. C1q is one of the factors responsible for the clearance of insoluble immune complexes, and its absence might contribute to the significantly high concentrations of insoluble immune complexes noted in individuals infected with HIV. (Procaccia, S., AIDS Vol 5, p. 1441 (1991)) Hypocomplementemia which characterizes HIV disease is correlated with HIV associated opportunistic infections and viral associated malignancies.
Regulators of complement activity can be found attached to plasma membranes. Others circulate freely in human plasma and lymph. Many regulators of complement activity (RCA) have been characterized and virtually every step in all three pathways is subject to positive and negative controls. Three enzymatic complexes (C3 convertases, C5 convertases, MAC complex) are focal within the complement cascade and are subjected to multiple inhibitors or catalysts.
Several proteins that control the complement activation pathways circulate in plasma as freely soluble molecules, and can either control C3 activation in the fluid phase or inhibit formation of MAC on cell surfaces. Regulators of complement, such as Factor H and low-molecular-weight Factor H-like proteins, have been shown to mediate this function. Factor H interacts with gp120, enhancing syncytium formation and soluble CD4 (sCD4) induced dissociation of the envelope glycoprotein (env) complex. Factor H only binds activated gp120 after it has engaged CD4, suggesting that the binding site is hidden within the env complex, and becomes exposed only after interaction of gp120 with CD4. (Pinter, C., AIDS Research in Human Retroviruses, Vol. 11, (1995)) The gp120 molecule binds to the CD4 receptor on helper T cells. The virus then fuses with the T cell. The fusion domain is located on gp41. Upon fusion, the gp120 fragment is shed. The gp41 ectodomain becomes exposed after shedding gp120. Binding sites for C1q and factor H on gp41 become unmasked.
HIV activates human complement systems even in the absence of specific antibodies. (Stoiber, H, J. Ann. Rev. Immunology, Vol. 15, 649-674 (1997)) This would result in viral inactivation if complement were unimpeded. The complement process if unimpeded would produce membrane attack complex (MAC), triggering virolysis. However, HIV avoids virolysis by incorporating into its structure various molecules of the host (e.g., DAF/CD55) that regulate complement. HIV includes these molecules in the virus membrane upon budding from infected cells, or by attachment to the gp41 and gp120 structures. (Stoiber, H., J. Ann. Rev. Immunology, Vol. 15, 649-674 (1997)) This interaction with complement components enables HIV to take advantage of complement components to enhance infectivity, follicular localization, and broaden its target cell range. At the same time, HIV defends against the humoral arm.
Proteins such as Factor H and CR1 have both cofactor and decay accelerating activities on the C3 convertases. (Stoiber, H, J. Ann. Rev. Immunology, Vol. 15, 649-674 (1997)) C3b integrity is essential for the complement cascade to culminate in cell lysis. C3b is rapidly cleaved by a serine protease (complement Factor 1-CF1) after interaction with appropriate complement receptors. Proteins that mediate this reaction possess cofactor activity for CF1. Some proteins down regulate complement activation by inhibiting the assembly and/or by favoring the dissociation of C3b and C4b generating enzymes (convertases). This activity is termed decay acceleration and is characteristic of the CD55 (DAF) protein molecule.
Serum lacking Factor H will lyse HIV and infected cells, but not cells that are uninfected. (Stoiber, H., J. Exp. Med., 183:307-310 (1996)) In the presence of Factor H, lysis of HIV has been shown to occur when the binding of Factor H was inhibited by a monoclonal antibody directed to a Factor H binding site in gp41. Human serum that is devoid of Factor H effectively lyses HIV virions. But to date, there has been no indication of how to implement this growing knowledge of the relationship of HIV and Factor H to the human complement.